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Ovarian cysts are linked to hormone imbalances and altered gene expressions, but the connection between cysts and ion channel expression is understudied. This study explored the role of TWIK-related acid-sensitive K + (TASK) channels in bovine ovarian cyst formation. The ovarian follicles were split into small (5 to 10 mm in diameter) and large (>25 mm in diameter) groups. Among the measured K + , Na + , and Cl - concentrations in follicular fluid (FF) obtained from small-sized follicles (SFs) and large-sized follicles (LFs), the K + concentration was significantly lower in LFFF. Quantitative PCR, Western blot, and immunocytochemistry data revealed that TASK-3 expression levels significantly decreased by approximately 50% in LFs and granulosa cells obtained from LFs (LFGCs) compared to the corresponding controls. The TASK-3 protein was localized to the plasma membranes of GCs. The diameters of LFGCs were larger than those of SFGCs. The cell swelling response to exposure to a hypotonic solution (200 mOsm/L) was highly reduced in TASK-3-overexpressing cells compared to vector-transfected cells. TASK-3-knockdown cells showed arrested growth. Senescence markers were detected in LFGCs and TASK-3-knockdown cells. These findings suggest that reduced TASK-3 expression in LFs is associated with the inhibition of GC growth, leading to senescence and cyst formation.

Follicular ovarian cysts (FOCs) are characterized by follicles in the ovaries that are >20 mm in diameter and persist for >10 days without the corpus luteum, leading to anovulation, dysregulation of folliculogenesis and subfertility in humans and livestock species. Despite their clinical significance, the precise impact of FOCs on oocyte reserve, maturation, and quality still needs to be explored. While FOCs are observed in both human and livestock populations, they are notably prevalent in livestock species. Consequently, livestock species serve as valuable models for investigating the molecular intricacies of FOCs. Thus, in this study, using goat FOCs, we performed integrated proteomic, metabolomic and functional analyses to demonstrate that oocyte maturation is hampered due to increased reactive oxygen species (ROS) in FOCs follicular fluid (FF) via downregulation of glutathione peroxidase (GPX1), a critical antioxidant seleno enzyme required to negate oxidative stress. Notably, GPX1 reduction was positively correlated with the FF's decline of free selenium and selenocysteine metabolic enzymes, O-phosphoryl-tRNA (Sec) selenium transferase (SEPSECS) and selenocysteine lyase (SCLY) levels. Adding GPX1, selenocysteine, or selenium to the culture media rescued the oocyte maturation abnormalities caused by FOCs FF by down-regulating the ROS. Additionally, we demonstrate that substituting GPX1 regulator, Insulin-like growth factor-I (IGF-1) in the in vitro maturation media improved the oocyte maturation in the cystic FF by down-regulating the ROS activity via suppressing Non-sense-mediated decay (NMD) of GPX1. In contrast, inhibition of IGF-1R and the target of rapamycin complex 1 (mTORC1) hampered the oocyte maturation via NMD up-regulation. These findings imply that the GPX1 regulation via selenocysteine metabolism and the IGF-1-mediated NMD may be critical for the redox homeostasis of FF. We propose that GPX1 enhancers hold promise as therapeutics for enhancing the competence of FOCs oocytes. However, further in vivo studies are necessary to validate these findings observed in vitro., Competing Interests: Declaration of competing interest The authors have no competing interests related to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Endometriosis (EM) is a gynecological condition known by the manifestation of endometrium alike soft tissue external to the usual place affecting up to 10% of all womenfolk in the reproductively active stage. However, the pathological process of endometriosis is not identified fully. The study aims to investigate the genes associated with the progression of endometriosis and its pathways using bioinformatics tools and techniques. The gene expression profile of three sets was retrieved, and bioinformatics data analysis was carried out for the microarray samples using GEO, DAVID, and STICH. Differently expressed genes (DEGs) refer to genes that exhibit significant changes in their expression levels between different conditions or groups, such as between different cell types, treatments, disease states, or developmental stages. DEG was determined based on a significant cutoff resulting in 298 unique elements based on the GEO Venn diagram map. DAVID (database for annotation, visualization, and integrated discovery) helps understand the biological significance of the data by identifying overrepresented biological terms, pathways, and functional annotations among a set of genes or proteins of interest. DAVID analysis revealed positively and negatively associated genes and followed by target proteins. DAVID is helpful for getting results of molecular mechanisms and pathways associated with DEGs. The gene expression studies showed that the m-RNA expression of all the genes was upregulated in the PA1 cell line. The present study identified five genes (COMT, CYP19A1, GALT, LTA, and STAR) from 298 unique DEGs using microarray data analysis, and 5 protein targets were also identified that were linked with EM. The study concludes that this information may provide a bridging gap in understanding the progression of endometriosis., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: Endometriosis is a heritable, complex chronic inflammatory disease, for which much of the causal pathogenic mechanism remain unknown.Despite the high prevalence of ovarian chocolate cyst, its origin is still under debate., Methods: Prevailing retrograde menstruation model predicts that ectopic endometrial cells migrate and develop into ovarian chocolate cyst. However, other models were also proposed. Genome-wide association studies (GWASs) have proved successful in identifying common genetic variants of moderate effects for various complex diseases., Results: A growing body of evidence shows that the remodeling of retrograde endometrial tissues to the ectopic endometriotic lesions involves multiple epigenetic alterations, such as DNA methylation, histone modification, and microRNA expression.Because DNA methylation states exhibit a tissue specific pattern, we profiled the DNA methylation for ovarian cysts and paired eutopic endometrial and ovarian tissues from four patients. Surprisingly, DNA methylation profiles showed the ovarian cysts were closely grouped with normal ovarian but not endometrial tissues., Conclusions: These results suggested alterative origin of ovarian cysts or strong epigenetic reprogramming of infiltrating endometrial cells after seeding the ovarian tissue. The data provide contributing to the pathogenesis and pathophysiology of endometriosis., (© 2024. The Author(s).)

The efficacy of human amniotic mesenchymal stem cell (hAMSC) ovarian injection in improving ovarian function in primary ovarian insufficiency (POI) patients has been shown in some reports. However, the safety and efficacy of hAMSC vein injection remains unclear. In this study, we evaluated the safety and efficacy of hAMSC intravenous injection in cynomolgus macaques and SD rats and provided evidence for clinical trials. The hAMSCs were transplanted three times in SD rats at low, medium, and high doses. The animal behavior and biochemical and biophysical parameters were routinely monitored on a 2-month period posttransplantation, and histopathologic examinations were also performed. Experiments on the acute toxicity, allergy test, and hemolysis test showed that hAMSCs possess good biocompatibility. Our results showed that the maximum tolerated dose of hAMSCs in SD rats was 4.0 × 10 7 cells/kg. The maximum safe dose with three injections of hAMSCs in SD rats was 5.0 × 10 6 cells/kg. In addition, the results demonstrated that hAMSCs may restore POI rat ovarian function after two injections of 2.5 × 10 6 cells/kg or 5.0 × 10 6 cells/kg, which improved the disturbed estrous cycle, hormone levels, and ovarian lesions induced by pZP3. In conclusion, the preclinical results suggested that the transplantation of hAMSCs may be safe and efficacious for SD rats at doses of 5.0 × 10 6 cells/kg and lower., (© 2023. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

Cystic ovary disease (COD) is a common cause of subfertility in dairy cattle. Therefore, the aim of this study was to provide novel concepts for cyst classification and to investigate the effects of COD on tubal microarchitecture, oviductal metabolic function, and the formation of the sperm reservoir. Bovine Fallopian tubes affected by follicular cysts, follicular cysts with luteinization and luteal cysts were investigated by a variety of microscopic and histological techniques and compared to control cows in metestrus and diestrus. We defined three types of cysts involved in COD, each of which had a characteristic wall thickness, inner wall appearance and cellular pattern within the cyst aspirate. Regarding the Fallopian tube, each cyst type was associated with a characteristic morphology, specifically the microarchitecture of the folds in ampulla, epithelial cell ratios, and ciliated/secretory cell size and form. Furthermore, each cyst type showed different patterns of tubal glycoprotein and acidic mucopolysaccharide synthesis, which was highly variable as compared to the controls. Our studies are the first to characterize the effects of COD on the Fallopian tube, which promotes the establishment of novel, cyst-specific therapeutic concepts in cattle and helps gain a holistic view of the causes of subfertility in cows with COD., (© 2024 The Authors. Molecular Reproduction and Development published by Wiley Periodicals LLC.)

The alteration of signaling molecules involved in the general metabolism of animals can negatively influence reproduction. In dairy cattle, the development of follicular cysts and the subsequent appearance of ovarian cystic disease (COD) often lead to decreased reproductive efficiency in the herd. The objective of this review is to summarize the contribution of relevant metabolic and nutritional sensors to the development of COD in dairy cows. In particular, we focus on the study of alterations of the insulin signaling pathway, adiponectin, and other sensors and metabolites relevant to ovarian functionality, which may be related to the development of follicular persistence and follicular formation of cysts in dairy cattle. The results of these studies support the hypothesis that systemic factors could alter the local scenario in the follicle, generating an adverse microenvironment for the resumption of ovarian activity and possibly leading to the persistence of follicles and to the development and recurrence of COD., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: The aim of the study was to investigate the existence of neuroendocrine cells and to compare the density of those in normal ovarian tissue, endometriotic and non-endometriotic benign ovarian cysts., Patients and Methods: Twenty patients with the diagnosis of endometrioma and 30 control subjects consisting of ovarian serous cystadenoma (n=10), ovarian mucinous cystadenoma (n=10) and normal ovarian tissue (n=10) were included. The tissues were prepared and assessed according to staining density by using the H-score method., Results: Tissues with mucinous cystadenoma were significantly more stained with PAS and VanGieson, when compared to women with endometrioma. Macrophage deposition was higher in cyst samples with endometrioma and in normal ovarian tissue when compared to serous cystadenoma and mucinous cystadenoma. Normal ovarian tissue was significantly more stained with PGP9.5, NSE and SYN when compared to endometrioma and non-endometriotic benign ovarian cyst. PGP9.5 staining was higher in normal ovarian tissue when compared with endometriotic lesions (p<.001). Endometrioma samples were significantly more stained with p53 when compared to non-endometriotic cysts and normal ovarian tissue. c-Kit staining was mild and not statistically significant among all groups., Conclusions: During endometrioma transformation, expression intensity of neuroendocrine markers decreases compared to normal ovarian tissue and other benign ovarian cysts.

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus. It affects many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, which limits early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared them to matched eutopic endometrium, unaffected endometrium and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell specific to the peritoneal lesions, with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesion microenvironments and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment that represents a comprehensive cell atlas of the disease in individuals undergoing hormonal treatment, providing essential information for future therapeutics and diagnostics., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment., (© 2021 The Authors. Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Background: More than a third of women could develop ovarian cysts during their lifetime. Jingshu granules are used for the treatment of gynecological disease of primary dysmenorrhea. However, the molecular mechanisms of Jingshu granules in ovarian cysts are still unreported. We aimed to find the active ingredients, molecular targets, and potential signaling pathways of Jingshu granules in ovarian cysts by using the systemic pharmacological analysis., Methods: Firstly, the effect of Jingshu granules on female hormones and reproductive organs of young female rats was evaluated. Secondly, candidate pharmaceutical ingredients of Jingshu granules were retrieved from the traditional Chinese medicine systems pharmacology (TCMSP) database and analysis platform. Potential protein targets for the active ingredients in Jingshu granules were then identified according to the oral bioavailability and drug-likeness indices. Thirdly, ovarian cyst-related gene targets were screened based on different databases. Finally, enrichment analysis was used to analyze the potential biological function of intersection targets between Jingshu granules and ovarian cysts., Results: In young female rats, Jingshu granules reduced the secretion of estradiol, progesterone, and prolactin and could affect the development of the uterus. This suggested that Jingshu granules played roles in hormone secretion and reproduction. From the TCMSP, a total of 1021 pharmaceutical ingredients of Jingshu granules were retrieved. After further screening, a total of 166 active ingredients and 159 protein targets of Jingshu granules were identified. In addition, 4488 gene targets of ovarian cysts were screened out. After taking the intersection, a total of 110 intersection targets were identified between potential protein targets of Jingshu granules and gene targets of ovarian cysts. In the functional analysis of 110 intersection targets, 8 signaling pathways including progesterone-mediated oocyte maturation (MAPK8 and CDK1 involved), GnRH signaling pathway (JUN involved), T cell receptor signaling pathway and Toll-like receptor signaling pathway (MAPK1 involved), NOD-like receptor signaling pathway (TNF, IL6, and IL1B involved), p53 signaling pathway (CDK2 and CDK4 involved), VEGF signaling pathway (MAPK14 involved), and PPAR signaling pathway (PPARG involved) were obtained., Conclusion: Our study revealed that Jingshu granules could function in patients with ovarian cysts through a number of molecular targets and signaling pathways. Our study may provide a new field into the mechanisms of Jingshu granules in ovarian cysts, from the molecular to the signaling pathway level., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Jili Xu et al.)

This study aimed to examine 25OHD 3 concentration in the fluid of follicular and follicular lutein cysts of sows in comparison with preovulatory follicles as well as immunolocalize vitamin D metabolic enzymes (CYP27B1 and CYP24A1) and determine their protein abundances in the cyst wall. We have shown for the first time that 25OHD 3 level in the fluid of both cyst types was significantly lower than in preovulatory follicles. Furthermore, we have demonstrated CYP27B1 and CYP24A1 protein immunolocalization and abundance in follicular and follicular lutein cysts. The abundance of protein for both metabolic enzymes was decreased in ovarian cysts when compared to preovulatory follicles. We propose that altered VD metabolism in ovarian cyst might associate with their formation in sows., (© 2020 Wiley-VCH GmbH.)

Background and Aim: While the relationship between obesity and reproductive dysfunction is well known, the physiological mechanism behind obesity-related infertility remains unclear. Previous work suggests that follicle development prior to ovulation is disrupted in obese individuals. Follicle-stimulating hormone (FSH) and anti-Mullerian hormone (AMH) are two key regulators of follicle development, and the poorest reproductive outcomes have been recorded when these hormones are imbalanced. In order to understand how obesity impacts the reproductive axis, the present study induces reproductive dysfunction in female rats using a high-fat, high-sugar diet (HFHS). Results: In our study, several animals on the HFHS diet displayed abnormal estrous cycles. The HFHS diet also resulted in an increased prevalence of ovarian cysts and decreased formation of corpora lutea. Across all groups, the FSH/AMH ratio displayed a strong negative correlation with pre-antral, antral, and total follicle counts. Moreover, rats on the HFHS diet displayed larger adipocytes and produced higher levels of leptin than controls. When combined with average adipocyte size in multiple regression, the FSH/AMH ratio was strongly associated with cyst formation in the ovary. Conclusions: These findings provide strong evidence for the potential relevance of a combined FSH/AMH ratio as a marker of ovarian health and follicular status. Therefore, this ratio reflects a complex interaction between the reproductive and metabolic systems.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

The objective of this study was to identify proteins that are differentially expressed in the cystic wall tissues of ovarian endometriotic cysts, simple ovarian cysts, and in normal ovarian tissues. Specimens of ovarian endometriotic cyst wall tissue, simple ovarian cyst wall tissue, and normal ovarian tissue (six specimens per group) were collected from patients who received gynecologic surgery, respectively. Differentially expressed proteins related to the ovarian endometriotic cysts were screened by use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with functional annotation and bioinformatics analyses. All differentially expressed proteins related to cysts were validated using immunohistochemistry methods in recurrent and non-recurrent ovarian endometriotic cyst. A total of 359 proteins were identified as up-regulated in ovarian endometriotic cyst groups when compared with both the normal ovary and simple ovarian cyst groups. The levels of 27 proteins were >two-fold higher in the ovarian endometriotic cyst group than that in the other two groups. Of note, the five most significantly upregulated proteins were Charcot-Leyden Crystal Galectin (CLC), Defensin, alpha 1 (DEFA1), S100 calcium-binding protein A9 (S100A9), S100 calcium-binding protein A8 (S100A8), and Ferritin Light Chain (FTL). Immunohistochemistry results showed that the changes of S100A9 and S100A8 were consistent with the results shown by iTRAQ. However, no similarity of CLC, DEFA1, and FTL proteins was found between iTRAQ and immunohistochemistry. The ratio of patients with abnormally high S100A9 and S100A8 expression in the recurrent ovarian endometriotic cyst group was significantly higher than that in the non-recurrent group (P < 0.05). Our data identify differentially expressed proteins S100A9 and S100A8, and suggest they may serve as novel molecular markers to predict postoperative recurrence of an ovarian endometriotic cysts. Abbreviations: iTRAQ: isobaric tags for relative and absolute quantitation; HPRD: Human Protein Reference Database; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EM: Endometriosis; COX-2: cyclooxyenase-2; NF-kB: nuclear factor kappa-B; PR-B: progesterone receptor type B.

The aim of this study was to investigate some of the growth and transcriptional factors originating from oocytes and granulosa cells in follicular fluid and to identify the relationships between the basic blood metabolite-metabolic hormones and intrafollicular lipopolysaccharide (LPS) concentrations. Thirty cows included in the study were allocated into 2 groups comprising 15 cows with healthy preovulatory follicles (cyclic cows) and 15 cows with confirmed cystic follicles. The ovaries and uteri of all cows were assessed by transrectal ultrasonographic examination. Blood serum samples were collected at 15, 25, 35, 45, and 55 d after calving for analysis of nonesterified fatty acids, β-hydroxybutyrate, insulin, glucose, IGF-I, ACTH, and cortisol. Ovaries and uteri were examined using transrectal ultrasound. Vaginal discharge was evaluated on the same days. Follicular fluid was also aspirated on days 35-55 from the healthy preovulatory follicles and cystic follicles using a transvaginal ovum pickup method. The densitometric levels of inhibin-α, growth and differentiation factor (GDF-9), bone morphogenetic protein (BMP-6), and GATA-4 and GATA-6 proteins were analyzed by the Western blotting technique; the concentrations of antimullerian hormone (AMH), IGF-I, estradiol-17 beta (E2), and progesterone (P4) were determined by ELISA; and the concentrations of LPS in the follicular fluid were measured by the Limulus amebocyte lysate test. The serum insulin, ACTH, and cortisol concentrations were higher in cystic cows than cyclic cows, but serum IGF-I concentrations were lower in cystic cows. The IGF-I concentrations of cystic follicular fluids were lower, whereas AMH levels were significantly greater than those of healthy preovulatory follicular fluids. The cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-α expression and LPS concentrations were significantly higher than in healthy preovulatory follicles. The proportion of pathologic vaginal discharge within 25 d postpartum in cystic cows were higher than in the cyclic group. In conclusion, it is suggested that intrafollicular dysregulation of the transforming growth factor-β superfamily, growth, and transcriptional factors is affected by high intrafollicular LPS concentrations and systemic metabolic changes and these disturbances may be responsible for the generation of ovarian cysts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Endometriosis is an oestrogen-dependent disease, and epithelial-mesenchymal transition (EMT) is involved in the process of endometriosis. Whether oestrogen could induce EMT in endometriosis remains largely unknown. Here, we reported that up-regulated expression of EMT markers in ovarian chocolate cyst is accompanied by high expression 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1), and exposure of primary human endometrial epithelial cells to oestradiol conditions could promote EMT occurrence and activate both β-catenin and Snail signalling. Furthermore, we found nuclear β-catenin and Snail expression was closely linked in ovarian endometriosis, and β-catenin knockdown abrogated oestrogen-induced Snail mediated EMT in vitro. This is due to that β-catenin/ TCF-3 could bind to Snail promoter and activate its transcription. These results suggested that β-catenin signalling functions as the Snail activator and plays a critical role in oestradiol-induced EMT in endometriosis., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Study Question: Are selective markers for the neuronal differentiation such as microtubule-associated protein 2 (MAP-2) and synaptophysin (SYP) as well as the nerve growth factor (NGF) expressed by fibroids, myometrium and eutopic endometrium?, Summary Answer: Neuronal markers NGF, MAP-2 and SYP are highly expressed in fibroids compared with matched myometrium, and this neurogenic pathway is upregulated by tumor necrosis factor (TNF) alpha in cultured smooth muscle cells (SMCs)., What Is Known Already: Uterine fibroids or leiomyomas are the most common benign tumors, accounting for approximately one-third of hysterectomies. The present trend is to improve the medical treatment avoiding surgery, also for fertility sparing; hence, the pathogenic mechanisms are investigated, aiming to develop new therapeutic strategy., Study Design, Size, Duration: This laboratory-based case-control study is focused on fibroids and myometrial specimens obtained between 2015 and 2017 from 15 women of reproductive age at the proliferative phase of the menstrual cycle. Leiomyomas, matched myometrium and endometrium from each woman were analyzed. Control endometrium was obtained from women undergoing surgery for ovarian cyst (n = 15)., Participants/materials, Setting, Methods: qRT-PCR, western blotting and immunostaining were applied to evaluate the expression of neurogenic markers; the effects of TNF on NGF, MAP-2 and SYP expression in cultured SMCs from leiomyomas and matched myometrium were analyzed., Main Results and the Role of Chance: qRT-PCR analyses using tissues from clinical patients showed that the levels of NGF, MAP-2 and SYP mRNA were significantly higher in uterine leiomyomas compared with their matched myometrium (P < 0.05), whereas only NGF was significantly increased in eutopic endometrium compared with healthy endometrium. In primary SMCs, isolated from fibroids or from the adjacent myometrium, NGF, MAP-2 and SYP mRNA expression were significantly increased by TNF treatment (P < 0.05). Finally, human endometrial stromal cells prepared from the endometrium of patients affected by uterine fibroids display higher TNF expression (P < 0.001)., Limitations, Reasons for Caution: qRT-PCR analysis and immunofluorescence validation are robust methods demonstrating a clear upregulation of neurogenic factors in leiomyomas, even though additional studies are needed to establish a correlation between increased neuronal gene expression and degree of pain, as well as the involvement of inflammation mediators in the development of the neurogenic unhinge. Therefore, more in vivo studies are needed to confirm the results achieved from primary cultured SMCs., Wider Implications of the Findings: The increased expression of neurogenic factors in uterine fibroids and endometrium may contribute to explain the painful stimuli. Accordingly, these neurogenic pathways may represent potential therapeutic avenues to treat the fibroid-related disorders., Study Funding/competing Interest(s): This study was supported by research grants from the University of Siena. The authors declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The lectin histochemical pattern (LHP) was characterized and compared in normal and cystic ovaries of sows. Six biotinylated lectins (PNA, SBA, WGA, RCA-1, DBA and UEA-1) were used on tissue sections. In the normal ovaries, the reaction to UEA-1 and SBA was mild to moderate in mesothelial and endothelial cells. RCA-1 staining was mild to moderate in theca interna of growing follicles, corpora luteum and mesothelium. In addition, this lectin presented strong reaction in endothelial cells, granulosa cells of atretic follicles, zona pellucida of growing follicles and plasma. DBA showed strong intensity in mesothelial and endothelial cells. There was mild to moderate reactivity to WGA in granulosa cells, corpus luteum and theca interna of follicles in development, and moderate in zona pellucida, in granulosa cells of atretic follicles and mesothelium. PNA staining was mild to moderate in oocytes and in the adventitia and media of medullary arteries. Changes in the LHP of the cystic ovaries were noted; however, there were no differences in these findings between the follicular and luteinized cysts. UEA-1 reactivity in the cystic ovaries was moderately reduced in the mesothelial and endothelial cells, whereas there was mild reduction in the DBA staining in the granulosa cells. Reaction to RCA-1 and WGA in the cysts also was decreased in theca interna, zona pellucida and granulosa cells of atretic follicles. Furthermore, endothelium and theca interna in the cystic ovaries presented mild reduction of marcation to SBA, whereas there was decreased reactivity to PNA in the oocytes and adventitia and media layers of the medullary arteries. The results of the current study show that cysts modify the LHP in swine ovaries. These changes of glycoconjugates in many ovarian structures could modify diverse process and may be one of the reasons for decreased fertility in sows., (© 2019 Blackwell Verlag GmbH.)

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1β, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1β, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1β and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1β and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Background: Endometrioma generally shows higher intracystic signal intensity (SI) than mucinous cystadenoma (MCA) on T 1 -weighted imaging (T 1 WI). Nonendometrioma-associated malignant ovarian epithelial tumors (nonendometrioma group) often show higher intracystic SI than benign tumors on T 1 WI, while the converse is true for endometrioma and endometrioma-associated malignant tumors (endometrioma group). However, it is sometimes difficult to differentiate between hemorrhagic and mucinous content based on SI on T 1 WI. Hemoglobin (Hb) and protein are both speculated to shorten T 1 ., Purpose: To examine MRI values and Hb and total protein (TP) concentrations in ovarian tumors., Study Type: Prospective., Specimen: In all, 182 samples from 167 cystic ovarian tumors., Field Strength: 1.5T, spin-echo T 1 WI, fast spin-echo T 2 WI., Assessment: The in vivo intracystic/psoas major muscle SI ratios were determined as references for intracystic SI. T 1 and T 2 values, cystic content inversion times (TIs), and Hb and TP concentrations were determined to evaluate differences between 1) endometrioma and MCA; 2) benign, borderline, and malignant tumors in the nonendometrioma group; and 3) those in the endometrioma group., Statistical Tests: Wilcoxon's rank-sum test, Kruskal-Wallis test., Results: In endometriomas (n = 43) and MCAs (n = 27), mean T 1 and T 2 (TP, Hb concentrations) were 428 and 162 msec (52.7, 12.00 g/dl) and 1639 and 600 msec (7.1, 0.06 g/dl), respectively (all, P < 0.0001). In the nonendometrioma group (epithelial benign, n = 56; borderline, n = 20; malignant, n = 25), these values were 1657 and 696 msec (6.4, 0.35 g/dl), 1235 and 400 msec (13.5, 0.83 g/dl), and 1184 and 311 msec (19.7, 0.84 g/dl), respectively (all, P < 0.0001). In the endometrioma group (endometrioma, n = 43; borderline, n = 3; malignant, n = 8), these values were 428 and 162 msec (52.7, 12.00 g/dl), 427 and 108 msec (16.6, 3.07 g/dl), and 1010 and 268 msec (24.2, 1.56 g/dl), respectively (all, P < 0.05)., Data Conclusion: TP and Hb concentrations were higher in the contents of endometriomas than MCAs, leading to lower T 1 and T 2 values. In the nonendometrioma group, TP and Hb concentrations were higher in the cystic contents of borderline and malignant tumors than benign tumors, leading to lower T 1 and T 2 values. Conversely, the cystic contents of borderline and malignant tumors in the endometrioma group showed lower TP and Hb concentrations compared to endometriomas, leading to higher T 1 and T 2 values., Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:1133-1140., (© 2018 International Society for Magnetic Resonance in Medicine.)

Study Question: Is endometriosis associated with aberrant sialylation patterns and what is the potential impact of such anomalies on cell migratory properties?, Summary Answer: The reduced α-2,6 sialylation patterns in the peritoneal fluid of endometriosis-affected women and in stromal and epithelial cells from endometriotic lesions could be associated with enhanced cell migration., What Is Known Already: Endometriosis is considered to be a benign disease although, like cancer, it has the characteristic of being an invasive disease with cells that have an enhanced capacity to migrate. Aberrant sialylation has been reported in various malignancies and it has been linked to tumour invasion and metastasis., Study Design, Size, Duration: We conducted a prospective laboratory study in a tertiary-care university hospital. We investigated non-pregnant patients who were <42 years of age (n = 273) when they underwent surgery for a benign gynaecological condition., Participants/materials, Setting, Methods: The study population consisted of 102 women with histologically proven endometriosis and 71 endometriosis-free controls, who underwent a complete surgical exploration of the abdominopelvic cavity. Peritoneal fluids were collected during the surgical procedures, and endometrial and endometriotic biopsies were performed on all of the patients to generate stromal and epithelial primary cell cultures. The expression of α-2,6-sialyltransferase (ST6GALNAC1) was studied in eutopic and ectopic endometria of endometriosis patients and in eutopic endometria of controls by reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR). The α-2,6 sialylation levels were measured by ELISA in the peritoneal fluids of patients and controls and by western-blot in primary endometrial and endometriotic cell cultures using Sambucus nigra agglutinin (SNA), an α-2,6 sialic acid-binding lectin. A transwell migration assay after incubation of the cells with neuraminidase was also performed to evaluate the impact of desialylation on eutopic endometrial stromal cell migration., Main Results and the Role of Chance: ST6GALNAC1 gene expression was significantly lower in endometriotic lesions compared to that in eutopic endometrium of endometriosis-affected patients and healthy endometrium (16-fold for both; P < 0.01). We observed a significant reduction in SNA levels in the peritoneal fluids of endometriosis-affected women compared to control women (median optic density (OD), 0.257; range, 0.215-0.279 versus median OD, 0.278; range 0.238-0.285; P < 0.01), as well as in stromal (mean OD, 705 907; standard error of the mean (SEM), 141 549 versus mean OD, 1.16 × 106; SEM, 107,271; P < 0.05) and epithelial (mean OD, 485 706; SEM, 179 681 versus mean OD, 1.25 × 106; SEM, 232 120; P < 0.05) ectopic endometriotic cells compared to control eutopic cells, indicating reduced α-2,6 sialylation. Finally, in the transwell migration assay, the eutopic endometrial cells of endometriosis patients migrated significantly more into the lower chamber after incubation with neuraminidase, indicating enhanced migration by these cells after desialylation., Large Scale Data: N/A., Limitations, Reasons for Caution: Our control group involved patients operated for benign gynaecological conditions (e.g. tubal infertility, uterine fibroids or ovarian cysts) which may also be associated with altered sialylation patterns., Wider Implications of the Findings: The hyposialylation pattern of endometriotic cells appeared to be associated with enhanced migratory abilities, which might contribute to the establishment of early endometriotic implants. Further research is needed to confirm these findings, as this could lead to new potential therapeutic targets for this complex disorder., Study Funding and Competing Interest(s): No external funding was received and there are no conflicts of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH-induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH-induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH-induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level., (© 2018 Blackwell Verlag GmbH.)

Endometriosis has been considered as an estrogen (E2)-dependent and progesterone (P4)-resistant disease. On the other hand, lysophosphatidic acid (LPA) has been suggested as a significant modulator of ovarian pathology, acting via both LPA levels and LPA receptor (LPAR) upregulation. Therefore, the objective of the present study was to evaluate LPA concentration as well as LPARs, autotaxin (ATX), and phospholipase A2 (PLA2) expression in ovarian endometriotic cysts and normal endometrium with correlation of the expression of E2 and P4 receptors in endometriotic cysts. The analyses were carried out using the tissues derived from 37 patients with ovarian endometriosis and 20 endometrial samples collected from women without endometriosis were used as a control. We found that ovarian endometriotic cysts are a site of LPA synthesis due to the presence of enzymes involved in LPA synthesis in the tissue. Additionally, when we compared endometriotic cysts versus normal endometrium, we were able to show overexpression of 3 from 6 examined LPARs and both enzymes responsible for LPA synthesis in endometriotic cysts. Finally, we found the correlations between LPARs, ATX, and PLA2 and the expression of E2 and P4 receptors in endometriotic cysts. Owing to the high LPAR2 and LPAR4 transcript and protein expression in endometriotic ovarian cysts and positive correlations of both these receptors with the PR-B and ERβ, respectively, those receptors seem to be the most promising predictors of the endometriotic cysts as well as the main receptors responsible for LPA action in the ovarian endometriosis.

Introduction: Data on the possible role of peritoneal fluid free radical-mediated oxidative damage in the pathogenesis of endometriosis still remains inconsistent. The aim of the study was to determine iron metabolism markers and their influence on oxidative stress arameters in the peritoneal fluid of women with endometriosis., Material and Methods: 110 women with endometriosis and 119 patients with benign ovarian cysts were included in the study. All visible peritoneal fluid was aspirated during laparoscopy from the anterior and posterior cul-de-sacs. under direct vision to avoid blood contamination. Haemoglobin, iron, total oxidative status, and total antioxidant status were measured using standard colourimetric kits., Results: Haemoglobin, iron levels, as well as total oxidative status values were significantly higher, whereas total antioxidant status values were significantly lower in the peritoneal fluid of patients with endometriosis, in comparison to the reference groups. No differences were observed in peritoneal fluid concentrations of all parameters measured in relation to the phase of the menstrual cycle., Conclusions: Peritoneal fluid of women with endometriosis is characterized by disrupted iron metabolism. This is most likely related to an increased number of erythrocytes in the peritoneal cavity of endometriotic women, which leads to a higher concentration of haemoglobin in this environment. Impaired iron homeostasis may have a significant influence on the pathophysiology of peritoneal endometriosis by the direct impact of haemoglobin derivatives and/or formation of the pro-inflammatory and pro-oxidative environment. Peritoneal cavity oxidative stress occurs predominantly in women in advanced stages of the disease.

High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5' adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In ovarian cystic follicles, molecular changes in the growing follicle may have a local action and contribute to anovulation and cystic formation. One of the candidate molecules that affect the steroid and gonadotropin signaling systems of cystic follicles is lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. To examine the molecular characteristics of bovine cystic follicles, we analyzed LPS concentration in follicular fluid of cystic follicles, and the expression of LPS receptors and steroidogenesis-related genes in granulosa and theca cells. Cystic follicles were categorized as estradiol (E2)-active (EACF) and E2-inactive cystic follicle (EICF). Overall, LPS concentration in follicular fluid of EACF and EICF was higher compared with healthy preovulatory follicles (POF). Expression of luteinizing hormone receptor (LHR) in granulosa and thecal cells was decreased in EACF and EICF compared with POF. Expression of CYP19 in granulosa cells of EACF and EICF was lower than POF. High expression of StAR in granulosa and thecal cells was observed in EICF. In granulosa cells, the expression of TLR4 and TLR2 mRNA was higher in EICF than other follicles. By contrast, higher expression of TLR2 in thecal cells was observed in EICF. Thus, high LPS concentration in follicular fluid of cystic follicles may be associated with the regulation of expression of steroidogenesis-related genes in granulosa and theca cells. These finding revealed the molecular characteristics of bovine ovarian cysts and possible involvement of LPS in the pathology of cystic follicle diseases., (Copyright © 2018. Published by Elsevier B.V.)

The new paradigm of mutations in chromatin-modifying genes as driver events in the development of cancers has proved challenging to resolve the complex influences over disease phenotypes. In particular, impaired activities of members of the SWI/SNF chromatin remodeling complex have appeared in an increasing variety of tumors. Mutations in SNF5, a member of this ubiquitously expressed complex, arise in almost all cases of malignant rhabdoid tumor in the absence of additional genetic alterations. Therefore, we studied how activation of additional oncogenic pathways might shift the phenotype of disease driven by SNF5 loss. With the use of a genetically engineered mouse model, we examined the effects of a hypomorphic Vhl 2B allele on disease phenotype, with a modest up-regulation of the hypoxia response pathway. Snf5 +/- ;Vhl 2B/+ mice did not demonstrate a substantial difference in overall survival or a change in malignant rhabdoid tumor development. However, a high percentage of female mice showed complex hemorrhagic ovarian cysts, a phenotype rarely found in either parental mouse strain. These lesions also showed mosaic expression of SNF5 by immunohistochemistry. Therefore, our studies implicate that modest changes in angiogenic regulation interact with perturbations of SWI/SNF complex activity to modulate disease phenotypes., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

In Drosophila ovary, germline stem cells (GSCs) divide to produce two daughter cells. One daughter is maintained as a GSC, whereas the other initiates cyst formation, a process involving four synchronous mitotic divisions that form 2-, 4-, 8-, and 16-cell cysts. In this study, we found that reduction in the level of NHP2, a component of the H/ACA small nucleolar ribonucleoprotein complex that catalyzes rRNA pseudouridylation, promotes progression to 8-cell cysts. NHP2 protein was concentrated in the nucleoli of germline cells during cyst formation. NHP2 expression, as well as the nucleolar size, abruptly decreased during progression from 2-cell to 4-cell cysts. Reduction in NHP2 activity in the germline caused accumulation of 4- and 8-cell cysts and decreased the number of single cells. In addition, NHP2 knockdown impaired the transition to 16-cell cysts. Furthermore, a tumorous phenotype caused by Sex-lethal (Sxl) knockdown, which is characterized by accumulation of single and two-cell cysts, was partially rescued by NHP2 knockdown. When Sxl and NHP2 activities were concomitantly repressed, the numbers of four- and eight-cell cysts were increased. In addition, Sxl protein physically interacted with NHP2 mRNA in ovaries. Thus, it is reasonable to conclude that Sxl represses NHP2 activity at the post-transcriptional level to promote proper cyst formation. Because NHP2 knockdown did not affect global protein synthesis in the germarium, we speculate that changes in NHP2-dependent pseudouridylation, which is involved in translation of specific mRNAs, must be intact in order to promote proper cyst formation., (© 2018 Japanese Society of Developmental Biologists.)

In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1-Cre-induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3-kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP-inducible inhibitory Smads, was up-regulated in the Dullard mutant ovaries. Tamoxifen-inducible Dullard deletion in the whole body using Rosa26-CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN-193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard-deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling., (© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Cystic ovarian disease (COD) represents an important cause of infertility in dairy cattle and is associated with multiple physiological disorders. Steroidogenesis, which is necessary to ensure normal ovarian functions, involves multiple enzymatic pathways coordinated by insulin and other proteins. We have previously shown that cows with COD have an altered insulin response. Therefore, in the present study, we evaluated further alterations in intermediates downstream of the PI3K pathway and pathways mediated by ERK as critical signals for the expression of steroidogenic enzymes in the ovaries of control cows and cows with spontaneous COD. To this end, we evaluated the gene and protein expression of pan-AKT, mTOR, ERK1/2, and steroidogenic enzymes by real-time PCR and immunohistochemistry. Steroid hormone concentrations were assessed at systemic and intrafollicular level. Results showed altered expression of intermediate molecules of the insulin signaling pathway, whose action might modify the synthetic pathway of steroidogenic hormones. Similarly, the expression of steroidogenic enzymes and the concentration of progesterone in serum and follicular fluid were altered. These alterations support the hypothesis that systemic factors contribute to the development and/or maintenance of COD, and that metabolic hormones within follicles such as insulin exert determinant effects on ovarian functionality in cows with COD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This observational study compares the ratio of serum anti-Mullerian hormone (AMH) to the total antral follicle count (AFC) (as a marker of AMH production per follicle) in the various phenotypes of women with polycystic ovary syndrome (PCOS) and isolated polycystic ovarian morphology (PCOM). Two hundred and sixty-two women were recruited. Women with PCOS were divided into four phenotypes based on the diagnostic inclusion criteria of oligo-anovulation (OA), hyperandrogenism (HA) and polycystic ovarian morphology (PCOM). These included Group A (OA + HA + PCOM), Group B (OA + HA), Group C (HA + PCOM) and Group D (OA + PCOM). A ratio of serum AMH to total AFC was calculated and expressed as the AMH/AFC ratio which was compared in the phenotypes of PCOS and isolated PCOM. The median AMH/AFC ratios in PCOS-A, PCOS-D, PCOS-C and PCOM were 1.5, 1.6, 1.2 and 1.1, respectively. There were significant differences in the groups compared [F(3, 238) = 6.14, p = 0.000)]. The ratios were significantly higher in the oligo-anovulatory phenotypes PCOS-A and PCOS-D than the PCOM (p = 0.004 and 0.002, respectively). There was no significant difference in the ratio between ovulatory phenotype PCOS-C and PCOM (p = 0.59). The role of androgens and LH in per-follicle AMH production remains limited. The findings support the hypothesis of a key role for AMH in the mechanism of anovulation in PCOS.

Ovarian endometrial cysts cause some kinds of ovarian cancer, and iron is considered as one factor of carcinogenesis. In contrast, hypoxia is associated with progression, angiogenesis, metastasis, and resistance to therapy in cancer. We investigated hypoxia-induced perturbation of iron homeostasis in terms of labile iron, iron deposition, and iron regulatory protein (IRP) in ovarian endometrial cysts. Iron deposition, expression of IRPs, and a protein marker of hypoxia in human ovarian endometrial cysts were analyzed histologically. The concentration of free iron and the pO 2 level of the cyst fluid of human ovarian cysts (n = 9) were measured. The expression of IRP2 under hypoxia was investigated in vitro by using Ishikawa cells as a model of endometrial cells. Iron deposition and the expression of IRP2 and Carbonic anhydrase 9 (CA9) were strong in endometrial stromal cells in the human ovarian endometrial cysts. The average concentration of free iron in the cyst fluid was 8.1 ± 2.9 mg/L, and the pO 2 was 22.4 ± 5.2 mmHg. A cell-based study using Ishikawa cells revealed that IRP2 expression was decreased by an overload of Fe(II) under normoxia but remained unchanged under hypoxia even in the presence of excess Fe(II). An increase in the expression of IRP2 caused upregulation of intracellular iron as a result of the response to iron deficiency, whereas the protein was degraded under iron-rich conditions. We found that iron-rich regions existed in ovarian endometrial cysts concomitantly with the high level of IRP2 expression, which should generally be decomposed upon an overload of iron. We revealed that an insufficient level of oxygen in the cysts is the main factor for the unusual stabilization of IRP2 against iron-mediated degradation, which provides aberrant uptake of iron in ovarian endometrial stromal cells and can potentially lead to carcinogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

The aim of this study was to determine whether endometrioma resection alters most commonly defined endometrial metabolites, lactate (Lac), N-acetylaspartate (NAA), creatine 1 (Cr1), creatine 2 (Cr2), and choline (Cho) during the window of implantation. Twenty patients with uni- or bilateral endometrioma and 7 patients having nonendometriotic benign ovarian cyst were included. Midluteal phase magnetic resonance spectroscopy analysis of eutopic endometrium was performed before surgery. Second spectrum of endometrium was obtained 3 to 5 months after laparoscopic endometrioma resection. Pre- and postoperative endometrial peaks of Lac, NAA, Cr, and Cho were measured in units and denominated in parts per million (ppm). Compared to preoperative peak values, significantly decreased NAA, Lac, and Cr1 signals were noted in patients undergoing endometrioma surgery. Nearly 5-fold decline in the NAA signal occurred after endometrioma surgery (1.94 ± 3.24 vs 0.37 ± 0.55). Likewise, 2.5-fold decline in Lac signals was noted after endometrioma resection (2.81 ± 2.64 vs 1.06 ± 1.88). Both uni- and bilateral endometrioma affected endometrium signals the same. The peak intensity of Cho, Cr1, Cr2, NAA, and Lac did not alter significantly after nonendometriotic cyst surgery. Endometrioma surgery straightens endometrial NAA, Lac, and Cr1 peaks, suggesting improvement in endometrial receptivity.

The four latent transforming growth factor-β (TGF-β) binding proteins LTBP1-4 are extracellular matrix-associated proteins playing a critical role in the activation of TGF-β. The LTBP1 gene forms two major transcript variants (i.e. Ltbp1S and Ltbp1L) that are derived from different promoters. We have previously shown the importance of LTBP1 in vivo by using three different Ltbp1 null mice that were either deleted for exons 1 and 2 (Ltbp1L knockout), exon 5 (Ltbp1ΔEx5), or exon 8 (Ltbp1ΔEx8). While the Ltbp1L knockout and the Ltbp1ΔEx8 are perinatal lethal and die of cardiovascular abnormalities, the Ltbp1ΔEx5 is viable because it expresses a short form of Ltbp1L that lacks 55 amino acids (Δ55 variant of Ltbp1) formed by splicing out exon 5, while lacking the Ltbp1S variant. Since only the Ltbp1ΔEx5 mouse is viable, we have used this model to address aspects of puberty, fertility, age-dependent reproduction, and ovary function. We report for the first time a function of LTBP1 in female reproduction. The Ltbp1ΔEx5 females showed impaired fertility associated with delayed sexual maturity (p = 0.0074) and ovarian cyst formation in females older than 40 weeks (p = 0.0204)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: Platelet-derived growth factor B (PDGF-B) and nestin have been suggested to be useful in the assessment of neoangiogenesis in malignant ovarian masses. We aimed to investigate a possible association of these markers with newly formed microcapillaries and perivascular cells in ovarian tumors., Material and Methods: Microvessel density (MVD) and pericytes were studied in 82 women with ovarian neoplasms, including 7 benign cysts, 7 borderline masses, 64 epithelial ovarian cancers and 4 other malignant ovarian tumors. Immunohistochemical staining included antibodies to CD34, PDGF-B and nestin., Results: Median values of CD34-positive and nestin-positive MVD were: 24,5 (range:17-32) and 21 (range: 12-31), respectively. No significant correlation between intratumoral CD-34 positive MVD and nestin-positive MVD was found. Benign and borderline lesions more frequently than malignant tumors displayed low or medium values of nestin-positive MVD (p = 0.01). Histological grading of malignant tumors was associated with nestin-positive MVD (p = 0.01). Nestin expression in tumor cells was not correlated with tumor grade or histological subtype. PDGF-B expression was found in tumor microves-sels in 72% of cases (59/82). High expression of PDGF in pericapillary cells was strongly associated with high expression of this marker in cancer cells (p = 0.007). Significant correlations between PDGF-B and nestin expression in malignant tumor microvessels were also found (p = 0.04). Nestin and PDGF-B expressions were strongly associated with high grade tumors when compared to low grade or benign masses., Conclusions: We conclude that the assessment of PDGF-B and nestin-positive MVD could be used to identify only highly active, angiogenic malignant ovarian masses, where tumor vasculature is formed.

Long noncoding RNA (lncRNA) has been recognized as a regulator of gene expression, and the dysregulation of lncRNAs is involved in the progression of many types of cancer, including epithelial ovarian cancer (EOC). To explore the potential roles of lncRNAs in EOC, we performed lncRNA and mRNA microarray profiling in malignant EOC, benign ovarian cyst and healthy control tissues. In this study, 663 transcripts of lncRNAs were found to be differentially expressed in malignant EOC compared with benign and normal control tissues. We also selected 18 altered lncRNAs to confirm the validity of the microarray analysis using quantitative real-time PCR (qPCR). Pathway and Gene Ontology (GO) analyses demonstrated that these altered transcripts were involved in multiple biological processes, especially the cell cycle. Furthermore, Series Test of Cluster (STC) and lncRNA-mRNA co-expression network analyses were conducted to predict lncRNA expression trends and the potential target genes of lncRNAs. We also determined that two antisense lncRNAs (RP11-597D13.9 and ADAMTS9-AS1) were associated with their nearby coding genes (FAM198B, ADAMTS9), which participated in cancer progression. This study offers helpful information to understand the initiation and development mechanisms of EOC.

Cystic ovarian disease (COD) is an important cause of subfertility in dairy cattle. Bone morphogenetic proteins (BMPs), mainly BMP2, BMP4 and BMP6, play a key role in female fertility. In this study, we hypothesized that an altered BMP system is associated with ovarian alterations contributing to COD pathogenesis. Therefore, we examined the expression of BMP2, BMP4 and BMP6 and BMP receptor 1B (BMPR1B) in the ovaries of animals with spontaneous or ACTH-induced COD, as well as during the development of the disease, in a model of follicular persistence induced by low doses of progesterone (at 5, 10 and 15 days of follicular persistence). Results showed changes in BMP2, BMP4 and BMP6 expression during folliculogenesis, in granulosa and theca cells in the COD groups, as well as at different stages of follicular persistence. Results also showed changes in BMPR1B expression in developing follicles in animals with COD, and at the initial stages of follicular persistence (P5). Comparison between groups showed significant differences, mainly in BMP4 and BMP6 expression, in granulosa and theca cells of different follicular categories. The expression of these BMPs also increased in cystic and persistent follicles, in relation to antral follicles of the control group. BMPR1B showed high expression in cystic follicles. Together, these results may indicate an alteration in BMPs, especially in BMP4 and BMP6, as well as in BMPR1B, which occurs early in folliculogenesis and incipiently during the development of COD, which could be a major cause of recurrence of this disease in cattle.Free Spanish abstract: A Spanish translation of this abstract is freely available at http://www.reproduction-online.org/content/early/2016/08/01/REP-15-0315/suppl/DC1., (© 2016 Society for Reproduction and Fertility.)

Objective: To study the prominent differences between endometriosis (EMT) cells derived from ovary, oviduct and endometrium, and to provided new ideas about the pathogenesis of endometriosis., Patients and Methods: From June 2010 to June 2015, 210 patients diagnosed with endometriosis were enrolled in our study. Patients were treated by laparoscopy or conventional surgeries in our hospital. Ovarian chocolate cyst and paired normal ovarian tissues, fimbriated extremity of fallopian and uterine cavity endomembrane tissues were collected, prepared and observed by microscope. PCR was used for amplification of target genes (FMO3 and HOXA9) and Western blot was used to evaluate FMO3 and HOXA9 expression levels., Results: In 95 cases, endometriosis cells were derived from oviduct epithelial. In 110 cases, endometriosis cells were derived from the endometrium, and in 5 cases it was derived from the ovary itself. FMO3 gene transcription and protein expression were higher in oviduct cells while HOXA9 gene transcription and protein expression were higher in endometrial cells. In 89 cases the endometriosis cells were derived from oviduct epithelial and in 113 cases endometriosis cells were derived from the endometrium. Protein levels indicated that endometriosis cells in 85 cases were derived from oviduct epithelial and in 116 cases were derived from the endometrium., Conclusions: A large number of ovarian endometriosis cells were derived from oviduct epithelial.

The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities., (© 2016 by the Society for the Study of Reproduction, Inc.)

Increased inclusion cyst formation in the ovary is associated with ovarian cancer development. We employed in vitro three-dimensional (3D) organotypic models formed by normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells to study the morphologies of normal and cancerous ovarian cortical inclusion cysts and the molecular changes during their transitions into stromal microenvironment. When compared with normal cysts that expressed tenascin, the cancerous cysts expressed high levels of laminin V and demonstrated polarized structures in Matrigel; and the cancer cells migrated collectively when the cyst structures were positioned in a stromal-like collagen I matrix. The molecular markers identified in the in vitro 3D models were verified in clinical samples. Network analysis of gene expression of the 3D structures indicates concurrent downregulation of transforming growth factor beta pathway genes and high levels of E-cadherin and microRNA200 (miR200) expression in the cancerous cysts and the migrating cancer cells. Transient silencing of E-cadherin expression in ovarian cancer cells disrupted cyst structures and inhibited collective cell migration. Taken together, our studies employing 3D models have shown that E-cadherin is crucial for ovarian inclusion cyst formation and collective cancer cell migration.

Ovarian cystic follicles are the sign of important causes of reproductive failure in numerous species. In this review, some morphological, endocrinological and clinical aspects of cystic follicles in women, cows, mares, sows and bitches are discussed. Follicular cysts are the consequence of the failure of a mature follicle to ovulate at the appointed time of ovulation in the estrous cycle. Although the etiology of follicular cysts remains unknown, this review examines the evidence about the role of endocrine signaling systems in the specific disease or syndrome in each of the species mentioned above. This review also describes, the changes in the pathways of endocrine mechanisms that would trigger disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. The knowledge of the morphological and endocrinological nature of cystic follicles in different species can provide relevant information to better understand specific diseases when it is integrally analyzed from the comparative medicine viewpoint.

Ovarian cancer is a highly malignant neoplasm with high mortality rates. Research to identify markers facilitating early detection has been pursued for many years. Currently, diagnosis is based on the CA 125 and HE4 markers, as well as the ROMA algorithm. The search continues for new proteins that meet the criteria of good markers A total of 90 patients were included in the present study, allocated into: group 1, ovarian cancer, with 29 patients; group 2, endometrial cysts, with 30s; and group 3, simple ovarian cysts, with 31. Following histopathological verification, the CA 125, HE4, and metalloproteinase 3 (MMP3) levels were determined and the ROMA algorithm was calculated for all patients. The mean concentrations of all determined proteins, CA 125, HE4, and MMP3, as well as the ROMA values, were significantly higher in group 1 (ovarian cancer) compared to group 3 (simple ovarian cysts). The highest significant differences for the CA 125 levels (<0.000001) and ROMA (<0.000001) values were observed in postmenopausal women. For HE4, statistical significance was at the level of p=0.00001 compared to p=0.002 for MMP3. For the differentiation between ovarian cancer and endometrial cysts, the respective AUC ratios were obtained for CA 125, HE4, and MMP3 levels, as well as the ROMA values ( 0,93 / 0,96 / 0,75 / 0,98). After removing the post-menopausal patients, the MMP3 AUC value for ovarian cancer vs. benign ovarian cysts increased to 0.814. For post-menopausal women, the MMP3 AUC value for ovarian cancer vs. endometrial cysts was 0.843. As suggested by the results above, both the CA 125 and HE4 markers, as well as the ROMA algorithm, meet the criteria of a good diagnostic test for ovarian cancer. MMP3 seems to meet the criteria of a good diagnostic test, particularly in postmenopausal women; however, it is not superior to the tests used to date.

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.

Objective: To evaluate the use of as an aid in the identification of women who can safely undergo conservative, non-surgical management., Methods: All patients referred to the Program in Women's Oncology for surgery with a pelvic mass are evaluated at a prospective multidisciplinary tumor board (TB) where ROMA and imaging are used for management recommendations. This study evaluated women presented to TB with a pelvic mass between 2009 and 2013 who had either surgical or conservative management., Results: Of the 498 patients assessed, 392 (79%) had benign disease, 22 (4%) had LMP tumors, 28 (6%) had stage I-II epithelial ovarian cancer (EOC), 36 (7%) had stage III-IV EOC and 20 (4%) had non-EOC. Using clinical assessment in conjunction with ROMA, the TB recommended observation in 188 (37.8%) women. All patients diagnosed with an invasive malignancy were recommended for surgery by the TB. In the 315 patients managed surgically, 212 were found to have benign disease and 84 women were diagnosed with an invasive malignancy. The sensitivity for the initial TB recommendations using ROMA in conjunction with clinical judgment for detecting malignancy was 100% with a specificity of 47.7% and a NPV of 100%. When including low malignant potential tumors the sensitivity was 99.1%. For stage I-IV EOC ROMA alone had a sensitivity of 95.3%., Conclusions: ROMA in conjunction with clinical assessment can safely identify women for conservative management., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The process of transformation of growing bovine follicles into cysts is still a mystery. Local expression of proteins or factors, including transforming growth factor β, growth factors, and transcription factors, plays a central role in mammals. Therefore, in abattoir-derived cystic ovarian follicles and follicular fluid, the role of some transforming growth factor β superfamily proteins, insulinlike growth factor-1 (IGF-1) and GATA-4 and GATA-6, were investigated. The relationship between intrafollicular lipopolysaccharide (LPS) and etiopathogenesis of ovarian cysts was also assessed. Data on the preovulatory follicle and the largest follicle (F1) were compared. The number of intrafollicular LPS-positive samples and LPS concentrations were higher in cysts. Immunohistochemical staining was mildly positive for IGF-1, inhibin alpha, and GATA-4 in thecal cells. Staining for anti-Müllerian hormone (AMH), growth differentiation factor-9, bone morphogenetic protein-6 (BMP-6), and GATA-6 was insufficient for their quantitation, and oocytes could not be stained for any of the proteins tested in the cystic follicles. Expression of BMP-6, inhibin alpha, and IGF-1 was moderately higher in granulosa cells of F1 follicles, and all the proteins were moderately expressed in granulosa cells in preovulatory follicles. However, loss of GATA-6 staining was significant in F1 follicles. Intrafollicular progesterone, IGF-1, and AMH concentrations in cysts and F1 follicles were significantly higher than those in preovulatory follicles. Western blot analyses revealed that follicular fluid inhibin-α was strongly expressed, whereas expression of growth differentiation factor-9, BMP-6, GATA-4 and GATA-6 was lower in cysts than in preovulatory follicles. Also, high intrafollicular AMH concentration and low BMP-6 expression were closely associated with cystic degeneration and atresia. In conclusion, immunohistochemical loss of BMP-6 and GATA-6 in the granulosa cells together with high intrafollicular LPS levels may play important roles in disruption of the ovulatory mechanism and steroidogenic reactions in type 2 cyst. Also, high intrafollicular AMH concentration along with low BMP-6 expression may be used as indicators of the bovine degenarative ovarian follicles., (Copyright © 2015 Elsevier Inc. All rights reserved.)